Chapter 7 Mononuclear Phagocytes in Immune Defense

• Macrophages: The ‘big eaters’. Macrophages are endowed with a remarkable capacity to internalize material through phagocytosis.

• Macrophages differentiate from circulating blood monocytes and are widely distributed throughout the body. Macrophages belong to the family of mononuclear phagocytes, which also comprise monocytes, osteoclasts, and dendritic cells. These cells share a common hematopoietic precursor that cannot differentiate into neutrophils. Phenotypically distinct populations of macrophages are present in each organ.

• Macrophages are highly effective endocytic and phagocytic cells. Macrophages have a highly developed endocytic compartment that mediates the uptake of a wide range of stimuli and targets them for degradation in lysosomes.

• Macrophages sample their environment through opsonic and non-opsonic receptors. Macrophages express a wide range of receptors that act as sensors of the physiological status of organs, including the presence of infection.

• Clearance of apoptotic cells by macrophages produces anti-inflammatory signals. Macrophages produce IL-10 and TGF-β upon internalization of apoptotic cells.

• Macrophages coordinate the inflammatory response. Recognition of necrotic cells and microbial compounds by macrophages initiates inflammation leading to the recruitment of neutrophils. Monocyte recruitment to sites of inflammation is promoted by activated neutrophils and there is a collaborative effort between macrophages and neutrophils to eliminate the triggering insult. Macrophages are actively involved in the resolution of the inflammatory reaction.

• There are different pathways of macrophage activation. TH1 cytokines such as IFNγ enhance inflammation and anti-microbial activity. TH2 cytokines induce an alternate activation that promotes tissue repair. TGFβ, corticosteroids and IL-10 can induce an anti-inflammatory phenotype.

Macrophages: the ‘big eaters’

Macrophages are cells of hematopoietic origin widely distributed throughout lymphoid and non-lymphoid tissues. They are endowed with a remarkable capacity to internalize material through phagocytosis, which makes them key players in both homeostasis and immune defense. Macrophages clear approximately 2 × 10¹¹ erythrocytes a day and are also implicated in the removal of cell debris and apoptotic cells, processes critical for normal development and physiology. The machinery mediating this homeostatic uptake also enables macrophages to recognize and internalize invading microorganisms, a process that facilitates clearance of infectious agents and elicits inflammation. Macrophages are highly heterogeneous and differentiate according to the environmental cues and physiological conditions present in tissues including the presence of microbes or cellular damage.

Macrophages differentiate from blood monocytes

Macrophages belong to the family of mononuclear phagocytes, which also comprise monocytes, osteoclasts, and dendritic cells. These cells share a common hematopoietic precursor that cannot differentiate into neutrophils or other cells of myeloid lineage. Monocytes circulate through the blood stream and are the precursors of macrophages in all tissues of the body, including secondary lymphoid organs even in the absence of an overt inflammatory stimulus. Monocytes may also develop into dendritic cells at sites of inflammation.

Monocytes are immune effector cells in their own right capable of detecting and internalizing pathogens and triggering inflammation. Recently, subpopulations of monocytes have been described in human and mouse blood. These subpopulations display differential expression of chemokine receptors and respond differently to stimulation indicating that they play distinct roles during inflammation with each subset implicated in either promotion or resolution of inflammation.

Within tissues the mononuclear phagocytes undergo maturation, adapt to their local microenvironment, and differentiate into various cell types (Fig. 7.1). Distinctive populations of resident macrophages are found in most tissues of the body; they differ in their life span, morphology, and phenotype, for example the microglial cells in the brain appear quite unlike mononuclear phagocytes in other tissues (Fig. 7.2). Resident cells have usually ceased to proliferate, but may remain as relatively long-lived cells, with low turnover, unlike neutrophils.

Fig. 7.1 Differentiation of mononuclear phagocytes

Mononuclear phagocytes and some dendritic cells differentiate from a precursor in the bone marrow. Differentiation into monocytes and tissue macrophages is driven by M-CSF. Inflammatory dendritic cells and Langerhans’ cells also appear to belong to this lineage although Langerhans’ cells may be directly bone-marrow derived. They are effective in presenting antigen to naive T cells. Some of the populations, indicated by a circular arrow, have some capacity for self renewal.

Fig. 7.2 Microglia in mouse brain stained for F4/80

Microglia are widely distributed in non-overlapping fields and show a dendritic morphology – mouse brain stained with antibody to F4/80.

(Courtesy of Dr Payam Rezaie.)

M-CSF is required for macrophage differentiation

Macrophage colony stimulating factor (M-CSF) is a major growth, differentiation, and survival factor, selective for monocytes and macrophages. It is produced constitutively by fibroblasts, stromal cells, endothelial cells, macrophages and smooth muscle. Most members of the mononuclear phagocyte family express the receptor for M-CSF, CD115 and mice deficient in M-CSF have major defects in different macrophage populations, including osteoclasts.

In contrast, granulocyte-macrophage colony stimulating factor (GM-CSF) primarily regulates myeloid cell production. It is only produced after cell activation and mice deficient in GM-CSF have no major defects in macrophage populations, with only the maturation of alveolar macrophage being altered leading to pulmonary alveolar proteinosis.

Macrophage populations have distinctive phenotypes

Some of the key features of resident macrophage populations in tissues are shown in Figure 7.w1. The study of the heterogeneity and distribution of mononuclear phagocytes has been made possible through the use of antibodies against differentiation markers. These markers can be located at the plasma membrane or endosomal compartments. In the mouse the F4/80 antigen and macrosialin (CD68) have proved useful in defining the distribution of mature macrophages in many (but not all) tissues. Differentiation antigens such as sialoadhesin, a lectin-like receptor for sialylated glycoconjugates, are particularly strongly present on populations of macrophages in lymphoid organs that do not express F4/80 or CD68. Macrophages expressing sialoadhesin are exposed to the blood and lymph and are thought to be involved in antigen delivery. In humans, the CD68 antigen, the human homolog of macrosialin, is widely expressed in macrophages while the F4/80 homolog EMR2 labels subsets of macrophages. Anatomical differences between mouse and human spleen correlate with different distribution of macrophage differentiation markers. The mannose receptor is expressed by macrophages in the red pulp of mouse spleen but is absent from these cells in human spleen.

Fig. 7.w1 Resident tissue macrophage populations

Resident tissue macrophage populations.

Comparing the differentiation markers expressed by macrophages in tissues with those expressed by macrophages differentially stimulated in vitro, allows researchers to propose hypotheses regarding the role of different macrophage populations in health and disease. For instance, alternatively activated macrophages were first characterized in vitro by studying macrophages treated with IL-4 in culture; the markers identified in IL-4/IL-13 treated cells together with additional markers discovered in the case of parasite infection are being used to investigate the acquisition by macrophages of an alternative activation profile in tissues. Interestingly, macrophages with characteristics of alternative activation are being detected during allergic responses, nematode infection and during wound healing. Differences in activation markers between human and mouse macrophages need to be taken into consideration when translating results obtained with preclinical models into the clinical setting.

Some of the important stimuli that modulate macrophage phenotype are listed in Figure 7.w2 and some of their characteristic cell surface receptors in Figure 7.w3.

Fig. 7.w2 Environmental influences on macrophages

Mediators that act on macrophages.

Fig. 7.w3 Macrophage 7tm G protein-coupled receptors for chemotactic stimuli

Macrophages express a variety of receptors for chemotactic agents. Fractalkine (CX3CL1) is unique as a transmembrane ligand – its receptor, CX3CR1, and CCR2 may be differentially expressed by monocytes, giving rise to inflammatory and resident macrophages.

The tissue environment controls differentiation of resident macrophages

It is possible to reconstruct a constitutive migration pathway in which monocytes become endothelial-like and line vascular sinusoids, as in the liver (Kupffer cells, see Fig. 2.3), or penetrate between endothelial cells. They underlie endothelia or epithelia or enter the interstitial space or serosal cavities (Fig. 7.3). The molecular mechanisms of constitutive macrophage distribution and induced migration are beginning to be defined, and involve cellular adhesion molecules, cytokines, and growth factors, as well as chemokines and chemokine receptors.

Fig. 7.3 Differentiation and distribution of macrophages

Blood monocytes are derived from bone marrow and enter tissues initially as extravascular macrophages or tissue macrophages which may subsequently migrate into the serous cavities. At sites of inflammation monocytes may develop into elicited inflammatory macrophages or dendritic cells which can migrate through afferent lymph to the local lymph nodes.White arrows indicate recruitment during steady state conditions. Red arrows denote recruitment during inflammation.

Mature macrophages are themselves part of the stromal microenvironment in bone marrow. They associate with developing hematopoietic cells to perform poorly defined non-phagocytic trophic functions, as well as removing effete cells and erythroid nuclei. In bone, osteoclasts, highly specialized multinucleated cells of monocytic origin, mediate bone resorption and their deficency leads to osteopetrosis.

Secondary lymphoid organs contain several distinct types of macrophages. These macrophages have been better characterized in the mouse (Fig. 7.4) and subsets involved in the clearance of apoptotic lymphocytes (tingible body macrophages) or presentation of naive antigens to B cells (subcapsular sinus macrophages) have been identified. Anatomical differences between human and mouse spleen, such as the absence of a well defined marginal sinus, correlates with phenotypical differences in splenic macrophages (see Fig. 7.4)

Fig. 7.4 Macrophages in secondary lymphoid tissues

Heterogeneity of macrophages in secondary lymphoid organs of mouse and human. (1) Red pulp of spleen stained for F4/80. Macrophages stain strongly positive. (2) Mouse spleen stained with antibody to sialoadhesin. The marginal metallophils of spleen are strongly sialoadhesin (CD169) positive. (3) Human spleen stained for CD68 and (4) immunofluorescence staining with anti-CD68 (green), and for the mannose receptor (red) indicates staining along sinusoids but most CD68+ macrophages lack the mannose receptor. Nuclei are stained blue.

((1) Courtesy of Dr DA Hume. (2) Courtesy of Dr PR Crocker.)

Macrophages can act as antigen-presenting cells

Although macrophages are often regarded as sessile cells, they readily migrate to draining lymph nodes after an inflammatory stimulus and become arrested there. They are therefore absent from efferent lymph and do not, as a rule, re-enter the circulation.

Q. Why is there no advantage for the immune system in the recirculation of macrophages, whereas recirculation of lymphocytes is a central element in immune defense?

A. Macrophages do not develop an immunological memory, and do not undergo the selective clonal expansion of antigen-stimulated lymphocytes that occurs in lymphoid tissues. Therefore there is no advantage in having macrophages return to the circulation from lymphoid tissues.

Macrophages, like dendritic cells, have all the machinery required for antigen processing and presentation of exogenous peptides and endogenous peptides on MHC class II and class I, respectively. Cross-presentation, a process by which peptides of exogenous origin are presented on MHC class I, also takes place in macrophages. While dendritic cells are uniquely suited for stimulating naive T cells in secondary lymphoid organs, macrophages present antigen in the periphery to activated (already primed) T cells. This interaction makes macrophages important effector cells during adaptive immunity (Fig. 7.5). The specialization of dendritic cells for antigen presentation correlates with a reduced degradative capacity that facilitates the generation of MHC-peptide complexes.

Fig. 7.5 Comparison of the functions of macrophages and dendritic cells

Comparison of the functions of macrophages and dendritic cells.

Macrophages act as sentinels within the tissues

Macrophages react to a wide range of environmental influences that help to fulfill their role as sentinels of the innate immune system (Fig. 7.6). The presence of cells within tissues with the potential to initiate inflammation through the release of cytokines and chemokines and to cause tissue damage through the production of reactive oxygen species requires control systems capable of downmodulating macrophage activation. One of these systems involves the molecule CD200L, which is an inhibitory receptor expressed by myeloid cells. CD200L inhibitory signaling is triggered through interaction with CD200 expressed by non-hematopoietic cells as well as macrophages. The CD200–CD200L interaction is important for the control of macrophage activation by other cells present in tissues.

Fig. 7.6 Macrophage phenotype and function

Macrophage phenotype and function are modulated by the cytokines shown left. And they can sense their environment via opsonic receptors for antibody (FcR) and complement C3b (CR3) and via pattern-recognition receptors. They are also negatively regulated via CD200 and SIRPα (signal regulatory protein-alpha). Macrophages secrete cytokines and eicosanoids (prostaglandins and leukotrienes) and may release reactive oxygen and nitrogen intermediates (ROIs, RNIs) at sites of inflammation.

Phagocytosis and endocytosis

Soluble compounds are internalized by endocytosis

Macrophages play a key role in the clearance of altered forms of lipoproteins (e.g. acetylated lipoproteins) or glycoproteins (e.g. asialylated mannosylated proteins or proteins bearing advanced glycan products), damaged or apoptotic cells, pollutant particles and microbes. For this purpose, macrophages have a highly developed endocytic compartment that mediates the uptake of a wide range of stimuli and targets them for degradation in lysosomes. Soluble compounds are internalized through fluid phase or receptor-mediated endocytosis (see below) leading to the generation of vesicles called endosomes. Endosomes mature by fusing with different endocytic vesicles from the early and late endocytic compartments and finally the endocytosed material is targeted to lysosomes. This endosomal trafficking is also characterized by a reduction in the luminal pH which facilitates the action of acid hydrolases and degradation of the endosomal content.

Large particles are internalized by phagocytosis

Phagocytosis involves the uptake of particulate material (>0.5 μm) after recognition by opsonic or non-opsonic receptors (see Fig 7.6), its engulfment through the generation of pseudopodia and the formation of phagosomes. Phagosomes follow a similar maturation process to endosomes through the fusion with components of the early and late endocytic compartments so that maturing phagosomes sequentially adopt characteristics of early and late endosomes; this process culminates in the fusion of phagosomes to lysosomes to form phagolysosomes (Fig. 7.7). Phagosomal maturation is accompanied by acidification of the lumen (from 6.1–6.5 in early phagosomes to 4.5 in phagolysosomes), which controls membrane traffic and has a direct effect on microbial growth. Other microbicidal mechanisms associated with phagosome maturation are the generation of reactive oxygen and nitrogen species and the presence of antimicrobial proteins and peptides.

Fig. 7.7 Phagocytosis mediated by opsonic receptors

(1) Pathogens, such as bacteria or fungi, are taken up by binding to opsonic receptors including the Fc receptor, complement receptors, and receptors for carbohydrate e.g. mannose receptor (MR) or dectin-1. (2) The particle is engulfed and the phagosome forms (3). Acidification of the phagosome follows as toxic molecules (reactive oxygen and nitrogen intermediates) are pumped into the phagosome. The marker CD68 is located in the phagosome membrane. (4) Lysosomes fuse with the phagosome, releasing proteolytic enzymes into the phagolysosome, which digest the pathogen. (5) On completion the membrane of the phagolysosome is disrupted. Antigenic fragments may become diverted to the acidic endosome compartment for interaction with MHC class II molecules and antigen presentation. The process induces secretion of toxic molecules and cytokines.

Host cells control the phagocytic activity of macrophages by displaying the ‘don’t eat me’ signal CD47. CD47 engages a receptor in macrophages called SIRPα that through its immunoreceptor tyrosine-based inhibitory motif (ITIM) motif inhibits the uptake process. CD47 expression by tumor cells has been proposed as an immunosurveillance escape mechanism.

Macrophages sample their environment through opsonic and non-opsonic receptors

Macrophages are endowed with a wide range of receptors that act as sensors of the physiological status of organs, including the presence of infection. These receptors can be categorized as opsonic or non-opsonic depending on their capacity to interact directly with the stimuli or their need for a bridging molecule such as antibody or fragments of the complement component C3, which act as opsonins.

Opsonic receptors require antibody or complement to recognize the target

Bacteria opsonized by C3 fragments or antibody engage complement receptors (CR) or Fc receptors (FcR). CR-dependent phagocytosis is not an automatic process but requires additional stimulation such as inflammation. Monocytes and macrophages express a range of receptors (CR1, CR3, CR4) for C3 cleavage products that may become bound to pathogens, immune complexes or other complement activators. The role of CR3 role in regulated phagocytosis has been well studied, and the mechanism of CR3-mediated ingestion differs strikingly from that mediated by Fc receptors.

Q. What other function does CR3 have, unrelated to its role as an opsonic receptor?

A. CR3 is an adhesion molecule, which also has a role in cell migration by binding to ICAM-1 on endothelium or fibrinogen in the extracellular matrix (see Fig. 6.13).

FcRs belong to the immunoglobulin superfamily (see Fig. 3.17). The best characterized FcR is CD64 (FcγRI), the high affinity receptor for IgG which signals through the common γ chain that contains an immunoreceptor tyrosine based activation motif (ITAM). The common γ chain is also used by some non-opsonic receptors that bind carbohydrates (see below) and it signals through the key kinase Syk. In humans other activating receptors for IgG are low affinity FcγRIIa (CD32) and FcγRIII (CD16), which require the recognition of immune complexes for inducing internalisation. IgG-opsonized material is readily internalized by macrophages and leads to the production of reactive oxygen species and cellular activation. The activating effect of ITAM-associated FcγRs is regulated by the presence of the inhibitory form of CD32 (FcγRIIb), which bears an ITIM.

The mechanism for ingestion of antibody-coated particles is distinct from that mediated by CR3 (Fig. 7.8). FcR-mediated uptake proceeds by a zipper-like process where sequential attachment between receptors and ligands guides pseudopod flow around the circumference of the particle. In contrast, CR3 contact sites are discontinuous for complement-coated particles which ‘sink’ into the macrophage cytoplasm. Small GTPases play distinct roles in actin cytoskeleton engagement by each receptor-mediated process.